Project Summary/Abstract Mesenchymal stem cell (MSC) therapy has shown tremendous promise for enabling heart tissue repair after ischemic injury. The therapeutic effects of stem cells are mediated by paracrine factors. Several studies illustrate that exosomes (EXO) derived from stem cells play a critical role in stem cell mediated therapy of ischemic myocardium via increasing angiogenesis. From the translational perspective, EXO have greater salutary therapeutic effects than whole cells: EXO have fewer potential adverse effects, less immune rejection, are more amenable to manipulation, and EXO cargo can be modified by preconditioning or genetic manipulation. GATA- 4, a cardiac transcription factor, promotes cardiac morphogenesis, extends cardiomyocyte (CM) survival and preserves cardiac function via regulating various bioactive molecules and activating cardiac protective miRs. Our published data indicate that MSC overexpressing GATA-4 (MSCGATA-4) increase MSC survival, protect native CM and promote angiogenesis, compared to vector-transfect MSC (MSCnull). EXO derived from EXO derived from GATA-4 overexpressing GATA-4 (ExoGATA-4) are more efficient than EXO from vector-transfected MSC (Exonull) in protecting CM from ischemic injury. In our pilot study, ExoGATA-4 are enriched with pro-angiogenic miRs, which regulate vascularization. ExoGATA-4 downregulate the expression of thrombospondin 1, a well-known endogenous inhibitor of neovascularization. The overarching goal of this project is to exploit effectiveness of GATA-4 conferred EXO than the ordinary EXO in repairable effects of ExoGATA-4. Our central hypothesis is that the ExoGATA-4 are enriched with pro-angiogenic miRs and proteins that are transferred into recipient cells and active multiple signaling pathways, leading to angiogenesis and cardiac repair. Three Specific Aims are proposed: Aim 1, to determine the role of miRs carried by EXO in response to GATA-4 transfection. Aim 2, to demonstrate if the transferred bioactive molecules play a critical role in ExoGATA-4 mediated angiogenesis. Aim 3, to test the hypothesis that EXO derived from GATA-4 overexpressing MSCs are more effective than that from vector-transfected MSCs in promoting angiogenesis and myocardial regeneration. The proposed studies are innovative because no previous study has systematically examined the effectiveness of ExoGATA-4, with particular focus on the efficacy of EXO mediated angiogenesis and ischemic heart repair following systemic administration. Intravenous injection offers the advantage of enabling repeated treatments without the stress of repeated intramyocardial injections. The proposed study is highly significant because it will evaluate the concept that the effectiveness of cellular therapy can be reproduced by cell-free EXO and will explore this new modality for future clinical application by intravenous administration. This project will be the first systematic effort aimed at developing a completely new strategy in use of EXO administration in lieu of cells, which would revolutionize cell therapy. The outcome will offer a transformative validation and guide translational research and putative therapies based on enhanced angiogenic properties of EXO from genome-edited MSCs. 1